kls martin training course „cssd process technician“ 5.9 · 2016. 9. 14. · kls martin...

Dr. med. Dipl. -Ing. Thomas W. Fengler www.cleanical.eu Chirurgie-Instrumenten-AG Berlin

KLS Martin

Training Course „CSSD Process Technician“

5.9.2016

Block II:

Microbiology

CLEANICAL®:

Dr. med. Dipl.-Ing. Thomas W. Fengler Chirurgie-Instrumenten-Arbeitsgruppe Berlin

CMP GmbH CLEANICAL gmbh


Block II Microbiology

1. Microorganisms

2. Bacteria

3. Virus

4. Prions

5. Protozoa

6. Fungi

7. Epidemiological definitions

8. Biofilms

9. Biological Indicators

10. Washing, Disinfection, Sterilisation


Microorganisms

• Microscopic living organism formed by a single cell of a group of cells

• Quantitatively the most numerous on the planet, present in air, soil and water. Estimated 1 trillion species.

• Indispensable for life on earth

• First living beings on earth, crucial for evolution of current biodiversity

• Currently still essential for maintenance of the biosphere and balance of all living organisms

• This balance changes when microorganisms become the cause for infectious diseases in humans


Archea

Cysts

Algae

Bacteria Virus Others

Enterococcus

Staphylococcus

Streptococcus

Mykobacteria

Candida HIV

Polio

Rota

Noro

Herpes

Fungi

Microorganisms: main groups

Protozoa Prions

Plasmodium



1. Microorganisms

2. Bacteria

3. Virus

4. Prions

5. Protozoa

6. Fungi


8. Biofilms




Bacteria

Candida albicans E.coli

Pseud. aeruginosa Staphylococcus aureus


TEM*-Magnification 8x104

* Transmission Electron Microscope (TEM)


TEM-Magnification 12x104


Bacteria: characteristics

• Large morphological and metabolical diversity, great adaptability

• Frequent in aquatic and terrestrial environments

• Grow in presence of oxygen (aerobic) as well as in its absence (anaerobic)

• Many can fixate CO2 and create biomass

• Some carry out photosynthesis

• Some can fixate atmospheric nitrogen


Bacteria: industrial use

• Manufacture of Vitamins (A, B2, B12)

• Generation of organical acids ( acetic acid, butyrate, formic acid)

• Biotechnological genetic manufacture of substances using bacteria (insulin, alpha-interferon, hepatitis vaccines, etc.)

• Breakdown of organic materials (composting, water purification)

• Food industry (yoghurt, sour milk, sauerkraut, etc.)


Bacteria: Structure of a microbial cell Cytoplasm Nucleoid

Cell wall Cytoplasmatic membrane

Cell wall

Mitochondria

Cytoplasmatic

membrane

Cytoplasm

Mitochondria

Ribosomes

Nuclear

membrane

Nucleolus

Nucleus


Bacteria: structure of a microbial cell

• Cell wall

• Protein structure

• Carrier of Antigenicity (triggers immune reactions)

• The cell wall provides protection and structural stability to the cell.

• Responsible for different colouring processes (Gram + and Gram -)


Bacteria: gram positive or negative

Peptidoglycan

(murein)

Cytoplasmatic

membrane

Cytoplasmatic

membrane

Peptidoglycan (mureine)

Periplasmatic space

External membrane

Gram+

(Staphilococcus aureus)

Gram-

(Escherichia coli)


Bacteria: structure of a microbial cell

• Cytoplasmatic membrane

• Semipermeable membrane

• Osmotic barrier for substances

• Contains transport and synthesizing components

• Cytoplasm

• All substances in the interior of a cell. Includes cytosols (gel) and organelles

• Carries the RNA

• Most cellular activities occur here


• Flagellum

• Lash-like appendage of certain prokaryotic cells

• Function as locomotive and sensory organs

• Different arrangement schemes, i.e. at one pole, both poles, tufts or all around

• Glycocalyx (bacterial capsule)

• Protects from external impacts (drought, heat)

• Should be destroyed by disinfecting solutions

• Bacteria with a glycocalyx are more resistant

Bacteria: Structure of a microbial cell


Bacteria: morphological types


Bacteria: reproduction and spores

• They reproduce through cell fragmentation

• At different temperature ranges, most in between 20-40°C

• Can create spores, isolating themselves from the environment to survive harsh conditions

• Spores can survive extreme temperatures and dissecation

• Aerobic spore producers: bacillus

• Anaerobic spore producers: chlostridium

• Careful! Do not mistake with fungal spores; these are a reproductive structure


Bacteria: reproduction

They prefer warm, humid and dirty (nutrient-rich) environments!

After 8 hours:

16.777.216 Organisms


Bacteria synthesize toxins

• Exotoxin

• is secreted through the cell wall from the inside

• The Pathogen works remotely; the pathogen is located in a wound and spreads its toxins inside the organism

• Endotoxin

• Located inside the cell, it is only released when the pathogen dies and its cell wall breaks, eg. through the use of antibiotics


Bacteria: human microbiota

• Resident microbiota:

• Germs permanently located on organism

• Depend on age, nutrition and general condition of person

• Certain protective function against pathogen germs

• Transient microbiota:

• “Approaching microbiota”

• Changing microbiota

• Can be disrupted by:

• Antibiotic therapy

• Corticoid therapy

• Metabolic diseases

• Cytostatic therapy


Bacteria: typical diseases

• Boils, abscesses

• Meningitis

• Osteomyelitis

• Wound infections

• Sepsis

• Endocarditis

• Rheumatic Fever

• Peritonitis

• Appendicitis

• Enteritis


Bacteria: typical germs

Staphylococcus aureus:

• Gram positive circular bacteria in form of heaps

• Normal microbiota in nose and throat area on 60% of all people

• Triggers purulent hair follicle and nail bed infections

• Can lead to abscesses, postoperative wound infections, pneumonia and serious general infection (sepsis)

• Transmitted by smear infection and droplet infection, present in 30%of population, specially hospital-staff

• Post-op wound infections are caused by not complying with hygiene measures

• MRSA is a Methicillin-resistant strain of S. aureus. These must be treated with reserve antibiotics, often leading to delays in treatment



S. aureus: which hygiene measures are being ignored?

• Incorrect use of mouth and nose protection

• Nose not covered

• Respirator is humid

• Respirator not present

• Insufficient hand disinfection

• Insufficient exposure time

• Forgotten completely



S. Epidermidis (“proceeding from the skin”):

• Normal human microbiota of skin

• With hygiene errors it can lead to infections related to vein catheter treatments (Wound infection of catheter entrance point or sepsis) or vessel prosthesis (deep wound infections which can lead to removal of prosthesis)



E. coli:

• Normal human microbiota of intestines in all humans

• Hygiene errors lead to smear infections and post-op wound infections

• Also can lead to urinary tract infections, often caused by the use of urinary diversions



Bacillus subtilis:

• Bacillus subtilis spores are used as test-germ for microbiological testing of sterilisation devices

• Bacillus sterothermophilus is very heat resistant and therefore used to test hot steam sterilisation devices


Bacteria: MDR, MDRGN, MRSA

Bacteria that are exposed to antibiotics can develop a resistance to specific substances, becoming harder to treat.

Multiple Drug Resistance (MDR) is antimicrobial resistance shown by a species of microorganisms to multiple antimicrobial drugs, such as antibiotics. Different degrees of MDR exist, therefore terms like extensively drug resistant and pandrug-resistant have been introduced.

Multidrug resistant Gram-negative bacteria (MDRGN) are resistant to multiple antibiotics and can create bacterial infections that are a serious threat to hospitalised patients.

Methicillin-resistant Staphylococcus aureus (MRSA) is a microorganism (bacterium) responsible for several very difficult to treat infections in humans. MRSA is a strain of S. aureus that has developed this particular resistance through natural selection and horizontal gene transfer. The infection is not more virulent than other S. aureus infections, but much harder to treat.


Bacteria: Tuberculosis (Tb)

• Infectious disease caused by Mycobacterium tuberculosis (MTB) that usually affects the lungs, leading to chronic cough with blood-containing sputum, fever, night sweats and weight-loss. Can be:

• Latent: people with latent Tb do not spread the disease. Only 10% of latent Tb becomes active and, without treatment, half of those die as a consequence

• Active: people with active Tb spread the disease through the air and suffer the symptoms

• Tb infections occur more frequently in patients who suffer from AIDS and in those who smoke


Bacteria: Gas gangrene

Infection caused by Clostridium perfringens bacteria that produces gas in tissues in gangrene. Gas gangrene can cause myonecrosis (muscle tissue death), gas production, and sepsis. Progression to toxemia and shock is often very rapid. It can easily be noticed by the large, blackened sores that form, as well as a degree of loud and distinctive crepitus caused by gas escaping the necrotic tissue.

By Engelbert Schröpfer, Stephan Rauthe and Thomas Meyer. -

Diagnosis and misdiagnosis of necrotizing soft tissue infections: three

case reports. Cases J 2008, 1:252. doi: 10.1186/1757-1626-1-252, CC

BY 2.0, https://commons.wikimedia.org/w/index.php?curid=6886224



1. Microorganisms

2. Bacteria

3. Virus

4. Prions

5. Protozoa

6. Fungi


8. Biofilms




Virus

• Acellular structure

• Intracelular obligate parasite (totally dependent on the host to complete its life cycle)

• Can be found in all environments

Size comparison between different

Virus, compared to an E. coli

bacteria.

Source: Kayser et al. Medizinische

Mikrobiologie, 12. Auflage, Georg-

Thieme-Verlag Stuttgart, 2010, S.

397)


Virus: reproduction

• Life cycle split in two:

• Extracellular: nucleoproteic structure called virion

• Inert (metabolically inactive)

• Unable to reproduce

• Intracellular: naked genetic material

• Can be DNA or RNA

• Induces the host to replicate the genetic information and to fabricate the virion components

Intracellular

Virion


Virus: incubation period

• The time the virus takes to incubate depends on the type:

• I.e. Influenza, mumps, measles or chickenpox take a few days to three weeks

• Hepatitis B or AIDS take much longer


Virus: coating

• Disinfecting solution “virucidal”

• Effective against coated virus and additionally against uncoated virus

Not coated Coated

• Disinfecting solution “restrictedly virucidal”

• Effective only against coated virus


Virus: adenovirus

• Adenovirus reproduces predominantly in the throat, conjunctiva and intestines of humans

• Transmission from person to person

• Incubation time 5 – 8 days

• Adenovirus creates cold-like symptoms in upper airways, conjunctives and gastroenteritis


Virus: rotavirus

• Transmission orally-faecal

• Incubation time 1 – 3 days

• Triggers vomiting and diarrhoea

• Also appears as nosocomial infection, specially at newborn units


Virus: Hepatits B, Hepatitis C

• Hepatitis B: coated DNA virus, family Hepadnavirus. Composed of a core (HBc) and a surface (HBs).

• Hepatitis C: also coated, but contain RNA, family Flavivirus.

Hepatitis virus enter the body through injuries in mucous membranes or skin (needle injuries!). From there they enter the bloodstream and into their target, the liver. By attacking the liver cells, its functions are altered, causing symptoms like icterus (secretion of bilirubin into the bloodstream, causing characteristic yellow skin tone) or cell death, leading to liberation of liver enzymes into the bloodstream. Incubation time of Hepatitis B is around 25 and 160 days. Only 5 to 10% of hepatitis cases lead to a chronic disease with liver cirrhosis or liver cancer in its final stadium.

¾ of Hepatitis C infections are not accompanied by any symptoms. However, 50% to 90% of Hepatitis cases become chronic. Late consequences can present in form of liver cirrhosis or carcinoma.

Vaccines exist for Hepatitis B, but not for Hepatitis C. HB contagion probability is around 30% (high) when wounded by an infected needle. In comparison, HC probability is around 3%.


Virus: human immunodeficiency virus (HIV)

HIV causes the acquired immuno-deficiency syndrome (AIDS). This disease is transmitted via unprotected intercourse with an infected person, shared use of infected injection needles or, in the medical sector from cut or sting wounds (to be considered also in the CSSD environment).

Disease normally starts unnoticed or with unspecific, flu-like symptoms, followed by a year long complaint-free phase. However, during this time, specific immune cells are attacked. Infected people are already infectious during this phase. Risk of transmission, i. e. from a needle is low (0,3%). Fully developed, the disease debilitates the body, causing extreme weight and memory loss. Secondary infections, such as tuberculosis or pneumocystic pneumonia are characteristic, as well as certain tumours, such as Kaposi sarcoma.

HIV is a sensible virus, being effectively eliminated by using thermal or chemical disinfection methods.



1. Microorganisms

2. Bacteria

3. Virus

4. Prions

5. Protozoa

6. Fungi


8. Biofilms




Prions

• Infectious agent composed exclusively of protein material (PrP: prion protein), folded in different ways and able to induce abnormal folding and the creation of prions in other proteins

• Leads to infections that are similar to viral diseases

• Cause of transmissible spongiform encephalopathies, such as BSE (mad cow disease) or scrapie (in sheep). Other diseases include Creutzfeldt–Jakob disease (CJD) and its variant (vCJD), Gerstmann–Sträussler–Schenker syndrome, fatal familial insomnia, and kuru

• Different from other microorganisms, as they do not contain nucleic acids

• Prion diseases can arise in 3 different ways:

• Acquired

• Familiar

• Sporadic


Prions: replication mechanism

• Heterodimer model: The normal PrPc is folded abnormally and the resulting PrPsc can induce these transformations in other proteins, then behaving in a similar way to a viral infection

• Fibril model: PrPSc exists only as fibrils, and its ends bind PrPC and convert it into PrPSc

• The replication method is important for the design of drugs to combat prions. Models predict that this drug would need to bind to fibril ends to stop their growth


Prions: Creuzfeld-Jakob disease

• Creuzfeld-Jakob-disease has been known for decades

• This variant was described in England in 1966 for the first time including its actual health policy implications


Prions: Variant Creuzfeld-Jakob-Disease (vCJK)

• This variant is thought to be transmitted from animals to human through contaminated food

• This disease creates irreversible damage to the central nervous system


Prions: Variant Creuzfeld-Jakob-Disease (vCJK)

Because of the strong resistance of Prions and the Danger of transmission by medical procedures (specially ENT, Eyes, Neurosurgery and orthopaedics), the Robert Koch Institute (RKI) decided to create, working together with experts, recommendations for disinfection and sterilisation of surgical instruments when suspicion of Creuzfeld – Jakob – Disease is justified (Bundesgesundheitsblatt 8/96 s. 282 – 283).

• The RKI created a “Task Force vCJK” in collaboration with the German Medical Association.

• In April 2002 the results were published in the Bundesgesundheitsblatt (can be found under www.rki.de)

• Thorough cleaning in a WD with highly alkaline cleaners and sterilisation at 134° C during 5 minutes.

http://www.rki.de/



1. Microorganisms

2. Bacteria

3. Virus

4. Prions

5. Protozoa

6. Fungi


8. Biofilms




Protozoa

• Eukaryotic, unicelular, with motility and without a cellular wall. Predator.

• Classifications depending on motility:

• Sporozoans

• Ciliates

• Flagellates

• Amoeboids

A number of protozoan pathogens are human parasites, causing diseases such as malaria (by Plasmodium), amoebiasis, giardiasis, toxoplasmosis, cryptosporidiosis, trichomoniasis, Chagas disease, leishmaniasis, African trypanosomiasis (sleeping sickness), amoebic dysentery, acanthamoeba keratitis, and primary amoebic meningoencephalitis (naegleriasis).



1. Microorganisms

2. Bacteria

3. Virus

4. Prions

5. Protozoa

6. Fungi


8. Biofilms




Fungi

• Chemo-organotrophs (no use of sunlight)

• Terrestrial environments

• Recycling of organic matter

• Quitine cell walls

• Unicellular or filamentous

• Classification:

• True Fungi: Zygomicota, ascomycota, basidiomycota, deuteromycota

• Moulds: Slime moulds and water moulds


Fungi: reproduction

• Reproduction by asexual or sexual spores (different from bacteria!!!) or asexual mycelial fragmentation

• Some fungi, such as Penicilium or Aspergilus may exchange genetic information parasexually via plasmogamy


Fungi: moulds

• Moulds are a type of fungus that grow in the form of multicellular filaments called hyphae

• Example: Aspergillus

• Cause aspergillosis

• It creates a carcinogenic poison

• One-time ingestion causes tissue damage

• Repeated ingestion can cause liver, stomach and kidney cancer


Fungi: moulds

• Aspergillus glaucus

• Green spores on food

• Aspergillus fumigatus

• Causes organ mycosis


Fungi

Over 100,000 species. Not all of them make us sick. They are hard to detect, create spores and are therefore dangerous

… others are especially dangerous!

Some taste well…

//commons.wikimedia.org/wiki/File:Oral_thrush_Aphthae_Candida_albicans._PHIL_1217_lores.jpg

//commons.wikimedia.org/wiki/File:2006-10-25_Amanita_muscaria_crop.jpg



1. Microorganisms

2. Bacteria

3. Virus

4. Prions

5. Protozoa

6. Fungi


8. Biofilms




Epidemiological definitions

• Infektionsschutzgesetz (German Infection Protection Law): regulates since the 1. of January 2001 the legal obligations for prevention and control of Infectious diseases on humans. The goal of this law is to prevent infectious diseases in humans, recognize infections early and impede their transmission. The type of infection and its transmission vectors are insignificant in this context



• Contamination: In hospitals we talk about contamination when microorganisms exist on or in living or inert matter. The presence of not pathogen microorganisms is considered contamination as well. Devices or objects that are not sterile are considered contaminated.



• Colonisation: presence (settlement) of microorganisms on skin or mucous membranes



• Infection: Infections occur when pathogenic germs penetrate the body and start reproducing. Depending on health condition of person and the properties of the germ, this can lead to an infectious disease

• Germs can penetrate the body through different routes or vectors, ie. :

• wounds

• body orifices

• instruments

• medication

• It is important to differentiate between endogen and exogen bacteria



• Infection source: origin of an infectious disease, i. e. a sick person, germ carrier (incubation phase), chronic carriers or inert reservoirs for germs (sewage, soil, dust, food), in the medical field, i. e. devices



• Once more, transmission types:

• Airborne droplet infection: contagion through tiny droplets of secretion, which proceed from the human airways, travel through the air and are inhaled in by other people

• Smear infection: indirect contagion through contact with objects that are contaminated with infectious secretions, i.e. saliva, urine or stool

• Contact infection: transmission from a sick person to a healthy person through direct body contact or touch by contaminated material or surfaces



• Nosocomial infections: Those infections which can be traced back to a medical measure in a clinical or hospital treatment



• Pathogen: microorganisms are differentiated in pathogens, which can cause disease, and not pathogens, which are harmless and can even be beneficial, ie. milk acid bacteria that transform milk to yoghurt.

• Pathogenicity: ability of a microorganism to trigger a disease.



1. Microorganisms

2. Bacteria

3. Virus

4. Prions

5. Protozoa

6. Fungi


8. Biofilms




Biofilms are…

• living association of microorganisms, i. e. bacteria, algae, fungi, protozoa

• probably the oldest living communities on earth (3,5 Billion old fossils)

• a very successful model, able to survive in almost any environment

• most common way in which bacteria organize

• often invisible (unobservable on hidden surfaces and only noticeable using microbiological indicators)

• a serious problem when living on our medical devices!


Biofilms: What is biofilm?

• Biofilms are formed by a thin slime layer (film), embedded with microorganisms

• In everyday life we perceive them as “Slime” or “coat”

A small universe: many different types of microorganisms live in a biofilm, often with different living conditions. The symbiotic relationship between different specialist individuals increases diversity inside the biofilm. Aerobic and anaerobic spaces, canals and venting structures exist. [Centers for Disease Control and Prevention Rodney M. Donlan: "Biofilms: Microbial Life on Surfaces"]


Biofilms: formation

• Biofilms appear when microorganisms colonize interfaces. These interfaces are also called “substratum”

• All interfaces can be colonized by biofilms:

• Between gas and liquid phases, i. e. on water surface

• Between liquid and solid phases, i. e. rocks in a river

• Between different liquid phases, i. e. oil drops in water

• However, biofilms are found predominantly in aqueous systems, either on the water surface or a interface with a solid phase


Biofilms: formation

1. Initial attachment

2. Irreversible attachment

3. Maturation I

4. Maturation II

5. Dispersion

[From: Looking for Chinks in the Armor of Bacterial Biofilms Monroe D PLoS Biology Vol. 5, No.

11, e307 doi:10.1371/journal.pbio.0050307


Biofilms: the invisible enemy

• As microorganisms are strongly fixated within the biofilm, a continuous liberation of contaminants to the surrounding medium is not to be expected. Biofilm monitoring through product control is therefore virtually impossible

• Punctual anomalies will be observed instead. A high germ load might appear suddenly and instantly disappear again. This will not be a regular or reproducible occurrence

• Sometimes these anomalies will occur directly after disinfection, a sign that the treatment has destabilised the biofilm matrix and now shear forces, i. e. pulsing flushing are spreading biofilm fragments


Biofilms: on medical products

• Staphylococcus aureus on the inside of a catheter

• The Biofilm, the sticky substance that surrounds the bacteria is formed by polysaccharides

• This film protects bacteria from antimicrobial substances, i. e. antibiotics

• Staphylococcus can induce severe wound infections

[Centers for Disease Control and Prevention's

Public Health Image Library (PHIL)]

http://en.wikipedia.org/wiki/Centers_for_Disease_Control_and_Prevention



http://phil.cdc.gov/phil/home.asp


Biofilms: infectious diseases

• More than 60% of infectious disease pathogens protect themselves from the immune system through the formation of a biofilm

• During biofilm maturation during dispersion phase, large chunks of bacteria separate and disperse. This creates a source of gems that can lead to chronical and reappearing diseases in patients and even the often deadly sepsis

• Biofilms have been associated to a number of infections:

• Wound infections

• Bacterial endocarditis

• Periodontitis

• Urethritis

• Prostatitis

• Caries

• Chronical middle ear infection in children


Biofilms: what is currently being investigated?

• AS the discovery and elimination of biofilm is connected to great difficulties, investigation focusses on the prevention of biofilms to begin with, concretely the first colonization step

• Catheters constitute a specially big problem:

• 5 – 10 % of patients get infected daily caused by bacterially contaminated catheters,

• When used over a longer time period (≥28 days) this increases to almost 100% of patients. Therefore much investigation is done on these products



• Medical products (in this case catheters) are equipped with miniature sound sources that “coat” the device with soundwaves (100 to 300 kHz) and create tiny vibrations on the surface. This impedes the initial attachment of planktonic (single free floating) bacteria. This was tested and found to be very effective against 4 types of bacteria

• Careful trap!: If too much energy is applied to the surface, bacteria get stressed and enter a “increased attaching” mode in order to create a protecting biofilm as fast as possible

Zadik Hazan, Jona Zumeris u. a.: Effective prevention of microbial biofilm formation on medical devices by low-energy

surface acoustic waves. In: Antimicrobial agents and chemotherapy. Bd. 50, Nr. 12, 2006, S. 4144–4152.



Coating Urinary Catheters with an Avirulent Strain of Escherichia coli as a Means to Establish Asymptomatic Colonization

Barbara W. Trautner, MD, Richard A. Hull, PhD, John I. Thornby, PhD, and Rabih O. Darouiche, Md; , Houston, Texas.

• Fight fire with fire: Catheters are coated with a biofilm of innocuous Escherichia coli HU2117 bacteria, which consequently “infect” the patients and coat the walls of their bladder with their biofilm

• This does not damage the patient, it does however prevent colonisation of more dangerous microorganisms and significantly reduces the number of difficult to treat UTI (urinary tract infections)



Brave new world:

• US specialists from the new investigation area “synthetic biology” modified a strain of bacteriophages (Virus that nest in bacteria) called T7, so that they cause their host to produce an enzyme that attacks bacterial cells as well as the biofilm structure

• If a E. coli biofilm is infected with theses phages, it practically driven to suicide. This method was tested and produces a biofilm reduction of 99.997%

Dispersing biofilms with engineered enzymatic bacteriophage Timothy K. Lu and James J. Collins

Harvard–MIT Division of Health Sciences and Technology,Cambridge, MA

Gamma-phages

(Photograph Vincent Fischetti

Raymond Schuch,

The Rockefeller University.)


Biofilms: external object associated biofilm infections

…is the microbial infection and colonisation of catheters, implants and medical instruments, i. e. difficult to wash endoscopes

• The increasing use of synthetic materials in medical engineering has caused an increase of the biofilm-issues

• Specially because of the affinity of some microorganisms for the surfaces made out of biomaterials, half of all nosocomial infections can be traced to surgical implants, i. e. vein catheters, artificial cardiac valves, joint prosthesis, peritoneal dialysis catheters, pace makers, etc

• The starting point for microorganisms is the skin of patients and hospital staff, contact of outlets and connectors with tap water and other sources from the environment


Biofilms: in endoscopes

Internal operational canal surface of a new endoscope

Internal operational canal surface of an endoscope after 600 uses

Presentation by Biotech Germande, Bernard Marchetti Centre

de Coordination de la Lutte contre les Infections Nosocomiales

du Sud Est

http://cclin-sudest.chu-

lyon.fr/antennes/auvergne/Auvergne_journee05/Marchetti.pdf


Biofilms: fighting biofilms

Brush Brush Brush again

(and again: BRUSH!!)


Biofilms: careful, trap!

• More than just protection from detachment, the biofilm matrix protects microorganisms from changing PH levels and disinfecting solutions

• Problems that arise from this:

• First, biofilms will not be detected with unsuited monitoring

• Second, a wrong security in the use of washing and disinfection processes might benefit microfilms

• Stress increases tendency of planktonic bacteria of attaching to surfaces

• Be sure to use the right detergent for biofilm removal


Biofilms: Water quality

• If water quality is not observed, biofilm build-up might occur

• Biofilms occur when microorganisms colonize interfaces. They accumulate predominantly in aqueous systems, either on the water surface or on the boundary with a solid phase

• A Biofilm is composed by a slime layer (film) in which microorganisms, i. e. bacteria, algae, fungi or protozoa are embedded, which to some extend produce the film themselves


Biofilms: water supply in hospitals

• Biofilms in the water supply of hospitals constitute a major health risk

• Chlorine (0.3 mg/L) and chlorine dioxide (0.2 mg/L) impede the formation of a biofilm. Treating the water with UV radiation significantly reduces biofilm formation potential

• A 730 day old biofilm is removed by chlorine dioxide in 70 days, by chlorine in 130 days and is not removed by UV radiation

• Nevertheless, water treatment with chlorine is not undisputed


Biofilms: requirements for water quality

• Recommendations by the German Commission for Hospital Hygiene and Infection Prevention (KRINKO, RKI), requirements for processing of flexible endoscopes:

• “The final rinsing step should be carried out by using water that has microbiological drinking water quality and is free from pathogen microorganisms”

• “Microbiological impeccable water can be achieved through the use of sterile water filters”


Biofilms: requirements for water quality

• Tap water rarely has microbiological drinking water quality caused by:

• Dead pipes

• Biofilm tearing

• Standing water

• Autonomous filters:

• High capacity (200 L per day)

• Reliable germ reduction

• Easy handling

• Price per litre is small compared to sterile water


Biofilms: in conclusion, avoid biofilms

• Biofilms are persistent. It is much easier to avoid them beforehand than to remove them!

• Minimise the time that the endoscope is contaminated!

• After washing and disinfection don’t re-contaminate!

• Provide germ-free water!



1. Microorganisms

2. Bacteria

3. Virus

4. Prions

5. Protozoa

6. Fungi


8. Biofilms




Biological indicators: definition

• Microbiological testing system with defined resistance against specified sterilisation process


Biological indicators: DIN EN ISO 11138

• The operator is responsible for planning “microbiological testing”, as well as their time limits and implementation

• Relevant manufacturer information must be taken into account

• Deviations must be documented and explained

• Results of testing must also be documented


Biological indicators: manufacturer information


Biological indicators: DIN EN ISO 11138

• The currently used test method are biological indicators, which consist of standardised solutions of microorganisms on testing devices. These are designed so that, when the microorganisms are eliminated, we can conclude that sterilisation has been successful

• They are sterilised with a test charge and then examined in a laboratory to check for surviving microorganisms

• When working with Steam Sterilisation, “bacillus stearothermophilus” bacteria is used. These are bacteria that can form spores


Biological indicators: examples


Biological indicators

After sterilisation, these strips are cultivated in an apropiate nutritional substrate for 7 to 10 days in order to verify elimination of testing germs.


Biological indicators: vials

• These are used for a rapid self-test on site

• After sterilisation, the colour tube inside the vial is broken

• Subsequently, the vial is cultivated in an incubator at 56-60°C (according to manufacturer)

• This process takes 24 to 48 hours, depending on the manufacturer

• An untreated vial is always provided as a control sample


Biological indicators

• With a microbiological test we can, strictly speaking, only determine effectiveness of sterilisation in the specific charge that we tested

• This test is no longer used as an external quality control, but internal sample and operating measure

• More conclusive: proof of sterility after expiration date of sterilised instruments, normally every half year

• The first place is occupied by “thermo-electrical tests”, as well as control of ventilation and steam penetration, i. e. Bowie-Dick-Test and air leaking



1. Microorganisms

2. Bacteria

3. Virus

4. Prions

5. Protozoa

6. Fungi


8. Biofilms




Washing, Disinfection, Sterilisation: decontamination

• Decontamination: elimination of dangerous soiling from a person, object or unprotected surface. This contamination can be biological, chemical or radioactive. Decontamination ends with a reduction of contamination to a level where it is considered “safe to handle”

• In the medical field, we talk about disinfection of cleanrooms, surfaces or medical products and devices

• In a CSSD, decontamination is regarded as the step previous to sterilisation


Washing, Disinfection, Sterilisation

• Washing

• All working steps, combining chemical and mechanical measures, that allow obtaining cleanliness in varying proportions, in order to achieve a state where the object is “safe to use”

• RKI requirements: <100µg Protein / Instrument



AUTOMATED MANUAL



Results of manual washing vary depending on person and day:

Limited reproducibility with temperatures < 40°C

Manual washing is

carried out 1 by 1!



Automatized and standardised washing and disinfection with washer-disinfectors (WD)



• Take into account:

• Water

• Energy

• Time

• Safety

• Washing performance

• Validation

• Cost



• Disinfection:

• Process that specifically kills or reduces the number of microorganisms in or on an object so that no infection risk can exist from it anymore. Usually, disinfection is required to reduce germs by 105 times

• i.e. :

• Chemo-thermal disinfection

• Chemical disinfection with Ultrasound

• A0 Value: EN DIN ISO 15883-1: The A0 value describes the energy required to kill microorganisms. It is defined as the expended energy (temperature in °C / time in seconds) in which the disinfection process has generated the desired lethality effect

• An A0= 60 (generally viewed as being an acceptable minimum), based on the formula, means 80 °C/60 sec or 90 °C/6 sec or 70 °C/10 min, etc.



• Sterilisation:

• Elimination of all forms of microorganisms, including spores, as well as deactivation of all virus, plasmids and DNA-fragments in substances or objects, so as to achieve the status of “sterility”

• The theoretical probability that a capable living microorganism remains on the “sterile” product must be smaller than one in a million products. This relationship is called “Sterility Assurance Level (SAL)”, “sterile” usually corresponds to a SAL level of 10-6


Thank you for your attention!

CLEANICAL gmbh Scharnhorststr. 3

10115 Berlin Germany

[email protected]

Dr. med. Dipl.-Ing Thomas W. Fengler

kls martin training course „cssd process technician“ 5.9 · 2016. 9. 14. · kls martin...

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